Analysis of Action Patterns of D-enzyme and Glucan Phosphorylase by Subsite Theory

Disproportionating enzyme (D-enzyme, EC 2.4.1.25) is a transglycosylase and its reaction involves the participation of more than two molecules of a substrate; E+2×Gn→Gn-i+Gn+i. The HPLC analysis of digests of maltooligosaccharides (G3-G7) showed that maltose is not formed in any case. The products from all substrates except G4 are those resulting from maltosyl transfer as the predominant reaction. Glucan phosphorylase(EC 2.4.1.1) has a rapid equilibrium-random Bi Bi mechanism involving the two kinds of substrate; E+Gn+G1P→E+Gn-1+Pi. Purified G3 is of poor primer ability, and the time course of the reaction shows an accelerating curve. By incorporating a sufficient quantity of β-amylase in the digests, the true rates of the G3-primed reaction could be determined from the linear time courses to give the K4 value of 9.3 mM. Other kinetic parameters for a series of maltooligosaccharides (G4-G8) were also determined in both the synthetic and the phosphorolytic directions. The reaction mechanisms of both enzymes are more complicated than the hydrolytic reaction of amylases and do not obey the simple mechanism of Michaelis-Menten type. We attempted to apply the subsite theory to the two enzyme reactions to analyze the characteristics of their action patterns. The two enzymes were isolated from a β-amylase-deficient variety of sweet potato.